Cycling of circadian rhythm proteins

描述（由申请人提供）：长期目标是了解昼夜节律（~24小时）节律的分子基础。这些节律是由大多数生物体的内源性时钟控制的，并在许多不同的生理过程中表现出来。生物钟功能紊乱与睡眠障碍以及其他病理（如肿瘤生长）有关。内源性生物钟的分子性质主要是通过对果蝇（Drosophila melanogaster）的研究确定的。这些研究表明，生物钟是由特定的基因（所谓的“生物钟基因”）组成的，这些基因的蛋白质产物在一天中的特定时间调节自身mrna的合成。由此产生的反馈循环驱动下游生理成分的循环，进而驱动行为和生理节律。然而，维持这种24小时周期的反馈回路的机制尚不清楚。此外，在某些时钟mrna（有时甚至是时钟蛋白）水平保持不变的条件下，节律通常得以维持，这表明翻译后调控的关键作用。果蝇生物钟与光线的同步也依赖于这种翻译后机制。我们假设，时钟蛋白的反馈活动必须循环以维持时钟功能，并且这种活动的循环是由蛋白质稳定性、核表达和抑制转录能力的时间控制驱动的。时钟蛋白的这些特征在很大程度上受到磷酸化的影响，但关键的调控步骤尚未确定。我们建议使用我们最近发现/生成的工具来剖析果蝇主要循环蛋白，周期（PER）和永恒（TIM）的翻译后调控。我们还将研究生物钟对光的反应，这在控制蛋白质稳定性方面有其基础。具体目的是：(1)确定一种新的tim等位基因行为表型的机制。我们已经确定了一种新的tim突变，它影响PER和tim的稳定性和核定位以及PER的磷酸化。这为我们提供了一个强大的工具来解决潜在的核表达机制，并确定亚细胞定位对磷酸化和稳定性的影响；(2)确定PER和TIM上新的磷酸化位点的功能意义。我们通过质谱分析确定了PER和TIM上新的磷酸化位点。我们将研究这些地点在上述过程中的作用；(3)确定TIM对光响应所需的激酶。通过小分子抑制剂筛选，我们已经确定了TIM被光降解所需的激酶类别。我们将确定所涉及的具体激酶。

DESCRIPTION (provided by applicant): The long-term goals are to understand the molecular basis of circadian (~24 hour) rhythms. These rhythms are controlled by clocks endogenous to most organisms and are manifest in many different physiological processes. Disrupted functioning of clocks has been associated with sleep disorders as well as other pathologies such as tumor growth. The molecular nature of the endogenous circadian clock was determined largely through studies done in the fruit fly, Drosophila melanogaster. These studies showed that the clock is composed of specific genes (so-called "clock genes") whose protein products regulate the synthesis of their own mRNAs at a specific time of day. The feedback loop thus generated drives the cycling of downstream physiological components which, in turn, drive rhythms of behavior and physiology. However, the mechanisms that maintain such feedback loops with a 24 hour periodicity are not understood. In addition, rhythms are often maintained under conditions where levels of some clock mRNAs, and sometimes even clock proteins, are held constant, indicating the critical role of post-translational regulation. Synchrony of the Drosophila clock to light also relies upon such post-translational mechanisms. We hypothesize that it is the feedback activity of clock proteins that must cycle in order to maintain clock function, and that the cycling of this activity is driven by temporal control of protein stability, nuclear expression and ability to repress transcription. These features of clock proteins are affected, to a large extent, by phosphorylation, but key regulatory steps have not been identified. We propose to use tools we have recently discovered/generated to dissect the post-translational regulation of the major cycling Drosophila proteins, period (PER) and timeless (TIM). We will also investigate the clock's response to light, which has its basis in the control of protein stability. Specific aims are to: (1) Determine the mechanisms underlying the behavioral phenotype of a novel tim allele We have identified a novel mutation of tim which affects the stability and nuclear localization of PER and TIM and the phosphorylation of PER. This provides us with a powerful tool to address the mechanisms underlying nuclear expression, and to determine the effect of subcellular localization on phosphorylation and stability; (2) Identify the functional significance of novel phosphorylation sites on PER and TIM. We have identified novel phosphorylation sites on PER and TIM through mass spectrometry analysis. We will investigate the role of these sites in the processes listed above; (3) Identify kinases required for the TIM response to light. Through a small molecule inhibitor screen, we have identified classes of kinase required for TIM degradation by light. We will identify the specific kinases involved.